Liver and IGF system

1.5.1. Liver as Central Organ in IGF Homeostasis

The liver plays a central role in the IGF homeostasis (Baruch, 2000) because it is the main source of circulating IGF-I, some IGFBPs and ALS. Within rat liver, the biosynthesis of individual components of the IGF system is attributed to different cell population, i.e. IGF-I, IGFBP-1 and ALS to hepatocytes, and IGFBP-3 to non-parenchymal cells (Scharf et al., 1995a; 1995b; 1996a; 1998; Arany et al., 1994;

Gentilini et al., 1998; Zimmermann et al., 2000). Interestingly, despite the wide distribution of the IGF-IR throughout the body, the IGF-IR expression is almost undetectable in hepatocytes, the cells with the highest levels of IGF-I expression (Caro et al., 1988; Hartmann et al., 1990). In contrast, presence of the IGF-IR has been demonstrated in non-parenchymal liver cells such as hepatic stellate cells (Brenzel and Gressner, 1996; Scharf et al., 1998), sinusoidal endothelial cells (Zindy et al., 1992;

Zimmermann et al., 2000) and Kupffer cells (Zindy et al., 1992). Moreover, production of the IGFs by these cells has also been observed (Pinzani et al., 1990; Zindy et al., 1992;

Scharf et al., 1998).

KCKC

SECSEC

hepatocytes hepatocytes HSC

HSC

Space

Space of of DisseDisse

ALS ALS IGFPB IGFPB--33

IGFIGF 150 kDa150 kDa

complex complex

Figure 12. The liver - central organ of the IGF system. It is the main source of circulating IGF-I, IGFBP-3 and ALS, which form 150 kDa ternary complex, the most abundant transport form of the IGFs in the circulation. Within rat liver, the synthesis of IGF-I, IGFBP-3 and ALS is strictly compartmentalized. IGF-I is produced mainly by hepatocytes. Nonparenchymal liver cells, i.e. Kupffer cells (KCs), sinusoidal endothelial cells (SECs), hepatic stellate cells (HSCs), also capable to synthesize and secrete IGF-I.

1.5.2. Updated Concept of Liver Fibrogenesis

Liver cirrhosis is a common sequela of chronic liver injuries from many causes, including viral infections (hepatitis B and C), alcohol abuse, drugs, helminthic invasions, metabolic diseases due to overload of iron and copper, autoimmune destruction of hepatocytes and bile duct epithelium, or congenital abnormalities. Hepatic fibrosis results from a distortion of the rates of synthesis (fibrogenesis) and degradation (fibrolysis) of ECM molecules (Friedman, 1993; Ramadori et al., 1998). This process in the liver is characterized by a three- to six-fold overall increase and deposition of the ECM components with their subsequent molecular reorganization resulting in an altered composition of fibrotic matrix. Advances in the isolation and characterization of liver cells, in conjunction with progress in molecular biology, have led to important new insights into the cellular basis of hepatic fibrosis.

LMF

Hepatocyte SEC

Activated HSC Extracellular Matrix

Figure 13. The cellular basis of liver fibrogenesis. Until now, it is believed that hepatic stellate cells (HSC) located in the space of Disse, also known as Ito cells, are the key effectors of hepatic fibrogenesis. With an ongoing hepatic injury these cells are believed to undergo activation with transdifferentiation from this quiescent, vitamin A-rich phenotype to myofibroblast-like phenotype with high proliferative capacity and ability to produce large amounts of extracellular matrix. However, in parallel with the process of their activation HSC undergo apoptosis. Recent data have demonstrated that resident hepatic myofibroblasts located mainly in periportal and pericentral areas within the liver are morphologically and functionally distinct from HSC. The major feature of these cells is that in contrast to HSC they are resistant to apoptosis and they definitely represent a second cell population involved in hepatic fibrogenesis.

Until now, it is believed that hepatic stellate cells, also known as Ito cells, are the key effectors of the fibroproliferative response in the liver (Friedman, 1993; 1999; 2000;

Ramadori et al., 1998). In normal liver, this cell population is distinguished by prominent intracellular droplets containing vitamin A and is considered as the primary storage depot for retinoids in the liver. Both in vivo with an ongoing hepatic injury as well as in vitro after plating on culture dishes, these vitamin A-rich cells undergo a phenotypic transition from a quiescent, vitamin A-rich phenotype to myofibroblast-like phenotype (activated HSCs). In contrast to quiescent HSCs, myofibroblast-like HSCs per se are

highly proliferative and can produce large amounts of ECM proteins (Friedman, 2000).

However, several independent groups have clearly demonstrated that HSCs undergo apoptosis both in vitro and in vivo (Saile et al., 1997; Iredale et al., 1998; Fischer et al., 2002; Taimr et al., 2003). Furthermore, upon activation, both HSCs and KCs acquire the ability to produce certain apoptosis-inducing ligands such as CD95L and TRAILs, which via cognate receptors induce apoptosis in HSCs (Fischer et al., 2002; Taimr et al., 2003). Therefore, it is hard to believe that dying cells are responsible for fibroproliferative process in the liver. Moreover, transdifferentiation of one clearly identified HSC to myofibroblast has never been shown in vitro. Thus, it appears likely that myofibroblast-like cells involved in hepatic fibrogenesis may also arise from another cell type within the liver. Recent data have demonstrated that activated HSCs and liver myofibroblasts (LMFs), despite their common features, represent morphologically and functionally different fibroblast populations. Moreover, it has also been found out that the ECM proteins fibronectin and type I collagen, deposited in a fibrillar matrix, are synthesized in higher amounts by LMFs than by HSCs, suggesting similar but not identical roles of these cells during fibrogenesis (Knittel et al., 1999a). Furthermore, HSCs and LMFs are present in normal and diseased livers in distinct anatomical compartments and respond differentially to tissue injury. Acute liver injury results in most exclusive increase in the number of HSCs, while in chronically injured livers both HSCs and LMFs are involved in fibrogenesis (Knittel et al., 1999b).

At present, the precursor pool of LMFs is not identified, in spite of very important clinical relevance. The precursors of these cells could be resident cells of the fibroblast lineage in the liver such as portal fibroblasts, periductal fibroblasts, vascular myofibroblasts,

„second layer“ cells or capsular fibroblasts. Portal fibroblast, residing under the normal conditions in the portal mesenchyme, can be responsible for periportal fibrosis.

Periductal fibroblasts, which constitute a distinct subpopulation of mesenchymal cells in the portal tract, have been suggested to proliferate and transdifferentiate in response to bile duct ligation, causing periductal, periductular and periportal „biliary“ type of fibrosis.

In schistosomiasis, vascular smooth muscle cells or vascular myofibroblasts situated in the wall of portal vein branches and portal arteries were thought to perpetuate to matrix-producing cells, thereby leading to periportal fibrosis as well. So called „second layer“

cells are myofibroblasts located around the centrolobular vein. They were suggested to cause typical „alcoholic“ type of pericentral fibrosis. Finally, capsular fibroblasts

detected in Glisson’s capsule can also be a potential source of ECM in the liver (Cassiman et al., 2002; Ramadori et al., 2002).

Recently it has been suggested that epithelial-mesenchymal transition (transformation or transdifferentiation) may play a role in fibrogenic organ remodeling. Indeed, to date there is evidence suggesting that in renal fibrosis myofibroblasts can derive from tubular epithelial cells by an epithelial to mesenchymal transition (Yang and Liu, 2001; 2002;

Strutz et al., 2002). Recent studies have shown that hepatocytes, which are epithelial cells, can also undergo transdifferentiation to the migrating fibroblast-like cells with mesenchymal phenotype under noxious stimuli (Pagan et al., 1995; 1997; 1999).

However, it is equally likely that in these cases proliferating LMFs simply replace apoptotic epithelial cells (Powell et al., 1999).

1.5.3. IGFs, PDGFs and Liver Fibrogenesis

The possible role of IGF-I in the pathogenesis of liver cirrhosis is obscure. It has been shown that in liver cirrhosis the IGF axis was severely disturbed. Patients with liver cirrhosis had reduced IGF-I, IGF-II and IGFBP-3 serum levels (Moller et al., 1995;

Scharf et al., 1996b), which were associated with adverse clinical outcome and complications of advanced cirrhosis such as malnutrition (Mendenhall et al., 1989), insulin resistance (Shmueli et al., 1996), impaired immunity (Mendenhall et al., 1997) and osteoporosis (Gallego-Rojo et al., 1998). Intriguingly, recent in vivo studies have demonstrated that exogenous IGF-I improved liver function and reduced oxidative liver damage and fibrosis in rats with experimental liver cirrhosis (Castilla-Cortazar et al., 1997). Alternatively, recent studies have shown that proliferation of HSCs and accumulation of type I collagen, the principal ECM protein, by these cells in vitro is stimulated in response to IGF-I (Scharf et al., 1998; Svegliati-Baroni et al., 1999;

Gentilini et al., 1998; 2000; Pinzani and Marra, 2001).Therefore, it is believed that due to its chemotactic, mitogenic and fibrogenic activity IGF-I is locally released during hepatic injury and triggers HSCs and possibly LMFs, thereby leading to their activation, proliferation as well as to collagen production and, finally, to perpetuation of fibrogenic response within the liver.

Interestingly, although PDGF shares many common features with IGF-I, it has totally different expression pattern in liver cirrhosis. Normal liver has almost undetectable level of PDGFs and their receptors, and healthy individuals have low serum PDGFs levels.

Conversely, in liver cirrhosis, hepatic expression and circulating levels of PDGFs are

considerably higher and positively correlate with the severity of disease (Pinzani et al., 1996; Zhang et al., 2003). Moreover, it has also been reported that blockade of PDGF receptor expression in vivo had beneficial effect in animals with liver cirrhosis (Borkham-Kamphorst et al., 2004). At present, however, it is obscure whether there is any pathophysiological link between different expression patterns of IGF-I and PDGFs in liver cirrhosis.

1.6. Aim of Study

Recently published data clearly demonstrate that there are functionally different fibroblast populations within the liver. Apart from HSCs, LMFs can be regarded as an essential cell type of fibroblast lineage involved in liver fibrogenesis. Clearly, detailed insights into the mechanism of myofibroblast proliferation in the liver will allow to identify new molecular targets and to develop new therapeutic modalities for more specific, effective, less harmful modes of treatment capable to cease a progression of liver cirrhosis. Some of these targets could be components of the IGF axis. Therefore, the purpose of the current work was to study the expression and regulation of the IGF axis components in rat LMFs, and the specific issues which were addressed in this work are:

1) to assess the capability of LMFs of expressing IGF-I and IGF-II;

2) to study the expression of receptors for the IGFs and to characterize their regulation;

3) to determine IGFBP species produced by LMFs and to elucidate their regulation at transcriptional, protein and posttranslational levels;

4) to evaluate mitogenic and fibrogenic effects of IGF-I in LMFs;

5) to determine the role of endogenous and exogenous IGFBPs in LMFs physiology;

6) to study a cross-talk between PDGFR and IGF-IR signalling systems in rat LMFs and to understand its implication for liver fibrogenesis.